Preparation of acid carboxypeptidase

ABSTRACT

A new enzyme, acid carboxypeptidase, whose significant characteristics are to give high activity at the low optimum pH, i.e., pH 1.5 to 5.5, and whose substrate specificity, molecular weight and other properties are entirely different from those of already known carboxypeptidase, is prepared by cultivating organism belonging to Aspergillus to obtain the crude enzyme solution and then purifying the above-obtained enzyme solution by one or more processes which include appropriately selecting and/or combining, of fractional precipitation, solvent precipitation, dialysis, various chromatographies, gel filtration. Said enzyme thereby obtained has the great advantage of enzymatically hydrolyzing protein and peptides within a low pH range where the danger of contamination is minimized.

United States Patent- Ichishima et al.

[ Feb. 29, 1972 54] PREPARATION OF ACID CARBOXYPEPTIDASE [72] Inventors:Eiii lchishima; Furnihiko Yoshida, both of Noda-shi, Japan [73]Assignee: Kikkoman Shoyu (20., Ltd., Noda-shi,

Japan [22] Filed: Apr. 8, 1969 [21] Appl. No.: 814,269

[30] Foreign Application Priority Data Dec. 9, 1968 Japan ..43/89525[52] US. Cl. ..195/66 R, 195/62 [51] Int. Cl ..C07g 7/028 [58] Field ofSearch ..l95/62, 63, 66, 68

[56] References Cited UNITED STATES PATENTS 3,063,911 11/1962 Tanaka etal. ..195/66 X 3,149,051 9/1964 Yoshida et a1. ..195/66 3,304,239 2/1967Zuber ..i95l62 3,492,204 1/1970 Koaze etal. ..l95/62X Primary ExaminerA.Louis Monacell Assistant Examiner-D. M. Naff Attorney-Cushman, Darby &Cushman [57] ABSTRACT A new enzyme, acid carboxypeptidase, whosesignificant characteristics are to give high activity at the low optimumpH, i.e., pl-l 1.5 to 5.5, and whose substrate specificity, molecularweight and other properties are entirely different from those of alreadyknown carboxypeptidase, is prepared by cultivating organism belonging toAspergillus to obtain the crude enzyme solution and then purifying theabove-obtained enzyme solution by one or more processes which includeappropriately selecting and/or combining, of fractional precipitation,solvent precipitation, dialysis, various chromatographies, gelfiltration. Said enzyme thereby obtained has the great advantage ofenzymatically hydrolyzing protein and peptides within a low pH rangewhere the danger of contamination is minimized.

1 Claims, 5 Drawing Figures This invention relates to a new enzyme,carboxypeptidase acid, and the preparation thereof.

The enzyme, carboxypeptidase, is known as including carboxypeptidases Aand B which are extracted from animal pancreas, and C from fruitsubstances. Such enzymes as representated by carboxypeptidases A and Bare known to react to liberate amino acids step by step from theterminal carboxyl of protein or peptide, and the optimum pH for thisreaction is about 7.5. On the other hand, the pH at which this occurs incarboxypeptidase C falls somewhat towards the acidic side with theoptimum of about 5.3.

The inventors have separated a new type of carboxypeptidase wherein theterminal amino acid is liberated within the pH range from 1.5 to 5.5,andwhose substrate specificity is entirely different from those of theknown types. This new enzyme was named as acid carboxypeptidaseaccording to the nomenclature for enzymes given in the Report of theCommission on Enzymes of the International Union of Biochemistry,published in 1961. And, said new enzyme can be produced by culturingmolds which belong to genus Aspergillus.

Referring to the diagrams hereto attached,

FIG. I shows the influence of pH on the activity of acidcarboxypeptidase;

FIGS. 2 through 4, the results of chromatography of acidcarboxypeptidase by means of weakly acidic cation exchange resin(Duolite CS-IOI) (FIG. 2), phospho-cellulose (FIG. 3) anddiethylaminoethyl-cellulose (FIG. 4), respectively; and

FIG. 5, gel filtration of acid carboxypeptidase by Sephadex G-75.

The optimum pH of said acid carboxypeptidase according to this inventionfalls more towards the acidic side than that of other known types ofcarboxypeptidase. For instance, the optimum pH of said enzyme in actingover such substrates as benzoxylcarbonyl-L-tyrosyl-L-leucine,benzoxycarbonyl-L- glutamyl-L-tyrosine, andbenzoxycarbonyl-glycyl-L-prolyl-L- leucyl-glycine are 3.5, 3.1 and 3.2respectively. In FIG. 1, curve A represents the pH-enzyme activityrelationship when the reaction was conducted in the reaction mixturecontaining 0.025 percent benzoxycarbonyl-L-tyrosyl-L-leucine assubstrate, 0.0025 percent crude enzyme preparation, and 0.005 M sodiumacetate-hydrochloric acid buffer, under the temperature of 30 C; andcurve B, the same relationship when 0.025 percentbenzoxycarbonyl-L-glutamyl-L-tyrosine as substrate, and 0.025 percentcrude enzyme preparation were used.

Here is described the substrate specificity of said acidcarboxypeptidase. For conveniences sake here, a carboxyl terminal (asreferred to C-terminal hereinafter) of substrate peptide chain will beexpressed by RXY where R stands for amino acids, peptides, or otheracyl-substituted amino acids or peptides; and X and Y, for L-aminoacids. Taking up carboxypeptidases A and B, the action of the formerhydrolyzes the peptide linkage between X and Y, when amino acids on Y-position of C-terminal of substrate having free carboxyl radical areeither neutral or acid amino acid residues, with the exception ofproline; and the latter shows strong activity as in the above case onlywhen amino acids on Y-position are basic amino acids. On the other hand,the activity of said acid carboxypeptidase according to this inventionis the most strongly influenced by amino acid on X. For example, whenthe amino acid residue-X present in the substrate would be aromaticamino acid residue, such as tyrosine or phenylalanine, the hydrolyticactivity of said enzyme is brought to the maximum. When X would be acidamino acid residue, such as glutamic acid, the activity is still high;but, when X would be leucine residue, then it somewhat decreases. Otheramino acid residue, e.g., glycine, valine or proline, may be used, butthe activity decreases markedly.

The hydrolytic activity of said acid carboxypeptidase on peptidases andamides is summarized comparatively in table I, in which the influence ofvarious amino acid residues of substrate on the action of said enzyme isshown. The activity of said enzyme is expressed in table I by therelative quantity of the enzymatic activity which can release aminoacids from various RXY (with benzoxycarbonyl-L-tyrosyLL-leucine as astandard), at 30 C., pH 3.5 for l min., by the action of said enzyme ata specific concentration so as to bring the extinction coefficient to2.0 at 280 me, unless otherwise noted.

TABLE I Comparison of the hydrolytic activity of purified acidcarboxypeptidase on peptides and amides Enzyme activity Substrate(R-X-Y) Enzyme unit a amino substituted peptides:

(I) X aromatic amino acid residue benzoxycarbonyl-L-tyrosyl L-leucinebenzoxycarbonyl-L-phenylalanyl-L-leucine"benzoxycarbonyl-L-phenylalanyl-L-tyrosine"acetyl-L-phenylalanyl-L-diiodo tyrosine" (2) X acidic amino acid residuebenzoxycarbonyl-L-glutamyl-L-phenylalunine"benzoxycarbonyl-L-glutamyl-L-tyrosine (3) X leucine residuebenzoxycarbonyl-L-proIyl-L-leucyl-glycine (4) X glycine residuebenzoxycarbonyl-glycyl-L-leucine benzoxyearbonyl-glyeyl-L-phenylalaninebenzoxycarbonyl-glycyl-L-tryptophane benzoxycarbonyl-glycyl-L-proline 0benzoxycarbonyl-glycyl-L-prolyl-L-leucylglycyl-L- prolinebenzoyl-glycyl-L-lysine 5) X valine residuebenzoxycarbonyl-L-valyl-L-glutamic acid (6) X proline residuebenzoxycarbonyl-glycyl-L-prolyl-L-leucine amides:

benzoxycarbonyl-L-tyrosyl-L-leucyl'amide"benzoxycarbonyl-L-tryptophanyl-phenylalanylamide"benzoxycarbonyl-glycyl-L-phenylalanyl'amide"benzoxycarbcnyl-glycyl-L-leucyl-amide"benzoxycarbonyl-L-alanyl-L-phenylalanyl-amide" tripeptides:

glycyl-glycyl-glycine L-alanylglycyl-glyeine L-leucyl-glycyl-glycineglycyl-glycyl-L-leucine dipeptides:

L-tyrosyl-L-leucine glycyl-glycine glycyl-L-leucine glycyl-L-asparticacid L-leucyl-glyeine peptides containing D-amino acid residue:benzoxyearbonyl-L-tyrosylD-leucine glycyl-D-aspartic acid 0 "Him 0 0000OOOO 000 The concentration of substrate is 5 l0"M, with the followingexception: I0"M in case of benzoyl-glycyl-L-lysine and 5XIO"M in case ofdipeptides and tripeptides.

"A portion of substrate becomes insoluble at pH 3.0.

It is an essential factor, as understood from the data in table I, thatthe carboxyl radical of the substrate must be free to cause the reactionof saidacid carboxypeptidase. Said enzyme can be identified as a type ofcarboxypeptidase, as it demonstrates there is no reaction with thesubstrates, whose carboxyl radicals are blocked by amides and the like,e.g., benzoxycarbonyl-L-tyrosyl-L-leucyl-amide,benzoxycarbonyl-L-tryptophanyl-L-phenylalanyl-amide,benzoxycarbonyl-glycyl-L- phenylalanyl-amide,benzoxycarbonyl-glycyl-L-leucyl-amide,benzoxycarbonyl-L-alanyl-L-phenylalanyl-amide, and the like. However,from the natural characteristics as shown above and hereinafter, it isobvious that said enzyme still differs from the known carboxypeptidasesA and B.

Furthermore, said enzyme differs completely from exopeptidases such asdipeptidase andtripeptidase in the mode of action. Precisely, it isimportant in hydrolysis of said enzyme that the amino radical ofa-position of amino acid residue-X, i.e., next to C-terminal Y, shouldbe blocked by acyl radicals or peptides. In other words, said enzymedoes not react with dipeptides (e.g., L-tyrosyl-L-leucine,glycyl-glycine, glycyl-L- leucine, 'glycyl-L-aspartic acid,L-leucyl-glycine), and tripeptides (e.g., glycyl-glycyl-glycine,L-alanyl-glycyl-glycine, l.- leucyl-glycyl-glycine,glycyl-glycyl-L-leucine).

It is also recognized in table I that hydrolysis by said enzyme is moststrongly influenced by amino acid residues in the X- position; but, theamino acid residues in the Y-position and its vicinity can give someinfluence as well.

The rate of hydrolysis by said enzyme likely increases to a greaterextent when a-amino radical of X-position is sub-' stituted bybenzoxycarbonyl radical rather than by acetyl radical. Also, as thesubstrate specificity of said enzyme is largely affected by the aminoacid residue of next to the C-terminal Y, i.e., of X, said enzyme actsslightly on the substrates whose C- terminal is basic amino acid, e.g.,benzoyl-glycyl-L-lysine, and still a very little on the substrates whichhave proline in its C- terminal, e.g.,benzoxycarbonyl-L-prolyl-L-leucyl-glycyl-L- proline. Another requirementbesides the above-mentioned for substrate of said enzyme is that aminoacid residue of substrate should necessarily be the L-form. Therefore,such substrate as benzoxycarbonyl-L-tyrosyl-D-leucine which have D-amino acid residues do not bring hydrolytic activity to said enzyme.

So far as described above, said enzyme difi'ers completely from bothcarboxypeptidases A and B in substrate specificity. And, it furtherdiffers in the following aspects:

The activity of said acid carboxypeptidase is not inhibited by metalchelating reagents such as ethylenediaminetetraacetate (0.05 M) ando-phenanthroline, which remove metals linked with enzyme protein bycoordinate bond. Therefore, it can be concluded that said enzymecontains no metal necessary for the catalytic action; and this is asignificant difierence which distinguishes said enzyme from othercarboxypeptidases whichcontain metals taking part in catalytic action.The known exceptions are carboxypeptidase C extracted from citrus fruitsand penicillium-peptidase B taken from micro-organisms belonging togenus Penicillium, whose activities are not affected by metal chelatingreagents. However, the difference is still found where the active pH ofsaid enzyme is considerably lower than that of the above two enzymes.Furthermore, it is mentioned in literature that the general propertiesof penicillium-peptidase B should be more like dipeptidases rather thanof carboxypeptidases.

Again, unlike usual endopeptidases, said acid carboxypeptidase isincapable of cleaving peptide linkage in proteins or peptides. Forinstance, when a reaction mixture of said enzyme and protein is treatedwith trichloroacetic acid to obtain the precipitate of high molecularprotein fraction, the trichloroacetic acid soluble fraction of thesupernatant filtrate contains only free amino acids, but not peptides asdecomposition product. Accordingly, said enzyme can be clearlydistinguished from acid proteinases or other similar endopeptidasesproduced by Aspergillus whose optimum pH for action falls around2.5-3.0.

Said acid carboxypeptidase is inactivated at 60 C. and over, and at pH7.0 and over.

The molecular weight of said enzyme is estimated to be about 100,000 bySephadex. The molecular weight of said enzyme is determined as 122,000according to Yphantiss procedure. The sedimentation constant of saidenzyme at zero concentration is estimated as 7.3 S. Compared with this,the molecular weight of acid protease produced from Aspergillus saitoiis 35,550; and those of carboxypeptidases A and B, 34,000 and 34,300respectively. Here again a difference of said enzyme from others isnoted.

From the above experimental facts, it is concluded that said acidcarboxypeptidase is a new type of enzyme, and it has not been known yet.

The activity of said acid carboxypeptidase is then assayed according tothe following methods:

One milliliter of reaction mixture consisting of a certainquantity ofsaid acid carboxypeptidase and 5X10 M ofbenzoxycarbonyl-L-tyrosyl-L-leucine dissolved in 0.05 M acetate buffer(pH 3.5) or 5X10" M of benzoxycarbonyl-L- glutamyl-L-tyrosine in buffersolution of pH 3.l, is incubated at 30 C. for 20 min. At the end ofreaction, it is held at 30 C. for 30 min. with the addition of 200 [LLof 0.3 M sodium hydroxide solution to inactivate residual enzyme. Suchalkali reaction mixture is neutralized by 200 [1.1. of 2.5 percentacetic acid, and thereafter, with the addition of 2.0 ml. of 0.5 Mcitric acid buffer (pl- 5.0) and 1 ml. of ninhydrine mixture which isprepared by Yemm-Cocking method [E. Cocking and E. W. Yemm: Biochem.1.58, Xll (1954)], it is heated in boiling water. After 15 min. ofheating, it is agitated violently while cooling with ice, and brought tothe volume of 5 ml. by adding aqueous solution of ethyl alcohol (2:1v./v.). And, finally the color yield by ninhydrin reagent is determinedby measuring the absorbency at 570 mp, that indicating the amount ofamino acid.

One unit of said enzyme activity is defined as the amount of enzymewhich can release 1 p.M L-leucine frombenzoxycarbonyl-L-tyrosyl-L-leucine as substrate, at pH 3.5 and at 30 C.for l min. When other substrates are used, the quantity of amino acidsreleased by enzyme reaction is determined as leucine equivalent andexpressed by relative enzyme unit. Leucine or other amino acids releasedas the reaction products can be detected by ninhydrin reagent afterdeveloping with a thin-layer chromatography by silica gel G asadsorbent, or by amino acid automatic analyzer.

The preparation of said acid carboxypeptidase is disclosed as follows:

Some of the molds belonging to genus Aspergillus which are used in thisinvention are: Aspergillus usamii, Aspergillus saitoi, Aspergillusniger, Aspergillus inuii, Aspergillus aureus, Aspergillus awamon',Aspergillus nakazawaii, Aspergillus oryzae, Aspergillus sajae, etc. Someexample of the strains of these molds are: Aspergillus usamii R-0635(ATCC 14331), Aspergillus saitoi R-38l3 (ATCC 14332), Aspergillus nigerNRRL 330, Aspergillus inuii R-363 l (ATCC 14333), Aspergillus aureus R-4523, Aspergillus aureus R-65l2 (ATCC 14334), Aspergillus awamoriR-3523, Aspergillus awamori 1AM 23.90 (ATCC 14335), Aspergillusnakazawaii R-6822Y, Aspergillus oryzae var. magnasporus A-l-5,Aspergillus sojae KS, and so on. However, the strains usedaccording tothis invention are not limited to these strains, or varieties andmutants thereof; but, all strains of molds belonging to genusAspergillus, which can produce acid carboxypeptidase, can be used inthis invention.

The cultivation of the above-named strains according to this inventioncan be performed either in solid medium or in liquid medium.

In the case of solid culture (koji method), the materials for mediumsuch as wheat bran, defatted soybean, and the like, are mixed withappropriate nutrients such as ammonium chloride, if necessary, andwater. The mixture is sterilized under steam pressure and is cooled.And, after cooling, seed culture is inoculated and mixed well. Then, theincubation is carried out at 25-40 C. for 50-90 hr.

At the end of incubation, 5-20 times volume of water is added, and it isheld for 60-70 min. and thereafter extraction of crude enzymepreparation is carried out under pressure. The extracted liquor isfurther run through filtration, by which suspended solids, spores andother foreign substances are removed. Again, the obtained filtrate iscooled to l-5 C., and percent ethyl alcohol (2.5-3.0 times volume) whichhas been cooled before the l-5 C. is added. The mixture is thoroughlyagitated and allowed to settle for 10 hr. or more.

The precipitate is lyophilized under 0.1 mm. Hg, and the crude enzymepreparation of acid carboxypeptidase is obtained. In place of ethylalcohol, acetone, methanol or lum.) Wheat bran, starch, glucose and soforth are used as carbon source of the medium; and as nitrogen sources,soybean, casein, meat extract and so forth as organic materials, andammonium chloride and other inorganic materials are used. Besides, suchingredient as phosphate or other inorganic salts can be optionally used.Conditions in process of submerge culture are adjusted to bring theactivity of the desired acid carboxypeptidase to its maximum, byselection of strains to be employed and other factors. In case, forinstance, Aspergillus usamii is employed, it is desirable to adjust pHof the medium to 3.0 to 6.0, and the temperature to 30 C. or thereabout;and the incubation is carried out for 50 to 100 hr.

Submerge culturing can be performed under stationary conditions,shaking, agitation, or aeration. But, in a large scale cultivation, itis more effectively performed under aeration- Y agitation.

After cultivation is over, cultured mash is filtered, and the filtrateis concentrated, if necessary, and adjusted to about pH 4.0. Then, afterthe second filtration, the filtrate is taken, as in the case with solidculture, for precipitation by alcohol and thereafter lyophilized toobtain crude preparation of acid carboxypeptidase.

The crude enzyme preparation as above-obtained is purified by properlyselecting and/or combining the methods such as salting-out,adsorption-elution by various ion exchange agents (e.g., Duolite CS-lOl,phospho-cellulose and diethylaminoethyl-cellulose), gel filtration tofractionate according to molecular weight, and others.

An example of the analyses of the product obtained is illustrated intable ll below in which the activity of acid carboxypeptidase obtainedby solid culture of wheat bran with molds belonging to genusAspergillus, is measured.

TABLE II Enzyme unit/solid culture product Strain (koji) g.

Aspergillus uraml'l' R0635 Aspergillu: .rajac KS The following examplesare used for the purpose of illustration only and do not limit theinvention therein. 7

EXAMPLE 1 Eight-hundred forty kilograms wheat bran was sprinkled with500 I. water and sterilized for 40 min. with pressured steam of 1.1kg./sq. cm. After cooling of above, 3 kg. seed culture of Aspergillussaitoi R-38 l 3 which had been pure-cultured separately was inoculated;and such was thoroughly mixed. it was then placed onto l,600 koji traysand incubated for about 50 hr. under the temperature kept between and 45C. The koji trays were transposed once every 2 days, when thetemperature of the culture rose.

At the completion of the above culture, the material was transferred toan extractor in which it was held for 60-70 min. with 3,000 I. wateradded. Then, the extraction was carried out under pressure of 60 kg./sq.cm. As the result, 2,400 l. of liquid, about percent of the added water,was obtained, and it was further filtered through a proper filtrationunit to remove suspended solids, spores and so on. The filtrate obtained thereafter was collected into a precipitation tank where it wascooled to l5 C. with brine and added with 2.5-3.0 times volume ofpercent alcohol which had previously been cooled to l-5 C. Such mixturewith alcohol was agitated thoroughly and held for more than 10 hr. toaccomplish the precipitation. Then the precipitate was further separatedthrough continuous filter and immediately after that, lyophilized under0.1 mm. Hg. The dried product was crushed from which 16 kg. of crudeacid carboxypeptidase preparation was collected. The enzymatic activityof this crude preparation was 3.14Xl0 enzymeunit per 1 g., and the yieldwas 60 percent.

EXAMPLE 2 The mixture of 90 kg. wheat bran, kg. defatted soybean and 40kg. ammonium chloride was sterilized, after water was added, underpressure of 3 kg./sq. cm. for 1 hr., and transferred to 20-kl. fermentorin which water was added to bring the total volume to 10 kl. Uponadjusting the initial pH to 5.5 and the temperature of the medium to 35C., it was inoculated with seed spores in suspension which had beenseparately pure-cultured by thoroughly mixing lOO g. seed culture ofAspergillus saitoi R-38l3 with 500 ml. sterile water. Then, theincubation was carried out under aeration and agitation at 35 C.

After 4 days of incubation, the cultured medium was filtered throughappropriate filter with filter aid. The filtrate was concentrated underreduced pressure of 35 mm. Hg, at 35 C. 1,500 1. liquid obtained as theresult was thereafter adjusted to pH 4.0 with inorganic acid ashydrochloric acid and further filtered. Then, following the steps as inexample 1, the precipitation by ethyl alcohol and lyophilization wereconducted, and 20 kg. crude enzyme in powder form was obtained. Theactivity of acid carboxypeptidase thus obtained was 7.8Xl0 unit per 1g., and the yield was 60 percent.

When salting-out by ammonium sulphate was conducted instead ofprecipitation by alcohol as in examples 1 and 2, the precipitation withammonium sulphate at 70 percent gave the highest yield to acidcarboxypeptidase, i.e., 60 percent.

EXAMPLE 3 Weakly acidic cation exchange resin, Duolite CS-lOl, (DiamondAlkali Co., U.S.A.), which was preliminary buffered with 0.001 M acetatebuffer, was packed in a chromatography column (2 cm. diameter) up to theheight of 40 cm. A 20-ml. crude enzyme solution dissolving 500 mg. crudeenzyme preparation as obtained in example 2 was applied to the column,and it was chromatographed. The chromatog raphy was carried out using a300-ml. mixing chamber with stirrer which was equipped on the passagebetween stock solution and the chromatography column, enabling theoperation to run continuously up to the point where pH of the eluatebecame the same as the pH of acetate buffer of 0.15 M stock solution,i.e., pH 5.2. Thereafter, fractions of each 5 ml. were collected.

The result of chromatography of acid carboxypeptidase by Duolite CS-lOlwas shown in FIG. 2. it was found that, while the fraction of acidproteinase (or, Aspergillopeptidase A) produced by strain of genusAspergillus was nearly completely adsorbed on the resin, causingtherefore slow elution, said enzyme according to this invention washardly adsorbed on and easily eluted from the resin. There was hardlyany acid proteinase contained in said acid carboxypeptidase fractionwhich was easily eluted. The recovery of said enzyme was about 50il5percent, with a slight deviation by each operation of chromatography.The enzyme fraction obtained above was then concentrated with collodionbag or taken for the second step of purification after dialysis against0.005 M acetate buffer (pH 4.0).

Duolite CS-lOl may be replaced by Amberlite CG-50 (Rohm and Haas,U.S.A.), by which a similar result can be obtained.

The second step of chromatography is to separate said acidcarboxypeptidase by phospho-cellulose (Brown, U.S.A. or Tohoku Pulp,Japan) which is cationic exchange cellulose. Phospho-cellulose which waspreliminarily equilibrated with 0.005 M acetate buffer (pH 4.0) waspacked in a chromatography column (2 cm. diameter), up to the height of50 cm. in the column, 100 ml. acid carboxypeptidase fractionated as perthe first chromatography was adsorbed, and the chromatography wasperformed with 0.05 M sodium chloride dissolved in 0.005 M acetatebuffer (pH 4.0), continuously through a 500-ml. mixing chamber.Fractions were collected by 5 ml. each.

The result of the above chromatography on phospho-cellulose isillustrated in FIG. 3. The recovery of said enzyme was about 60 percent.Said enzyme fractions were collected and dialyzed against 0.005 Macetate buffer (pl-l 5.0), and thereafter taken either to the next stepof purification or to concentration for preservation.

The third step of chromatography was carried out using diethyl amineethyl cellulose (Brown, U.S.A.) which is anionic exchange resin.Diethyl-aminoethyl-cellulose equilibrated with 0.005 M acetate buffer(pH 5.0) was packed in a chromatography column (2 cm. diameter) up tothe height of 50 cm. Upon said resin in the column, 194 ml. acidcarboxypeptidase obtained by the preceding chromatography was adsorbed,and the chromatography was carried out introducing sodium chloridedissolved in 0.005 M acetate buffer (pH 3.0), continuously through a500-ml. mixing chamber. Fractions were then collected by 5 ml. each.

The result of the above chromatography with diethyl aminoethyl-cellulosewas illustrated in FIG. 4. Recovery of said carboxypeptidase was about60 percent. Fraction obtained was chromatographed again as before.Recovery thereafter was about 80 percent. Then, the active fractioncollected was dialyzed and concentrated for preservation.

The fourth step of purification was gel filtration on Sephadex G-75(Pharmacia, Sweden). The column size was 2 cm. in diameter and 65 cm. inheight. Sephadex G-75 was equilibrated with 0.01 M acetate buffer beforebeing applied to the column. The lyophilized enzyme preparation obtainedabove was dissolved in 5 ml. acetic acid solution and poured on to theupper portion of the column where gel filtration was performed with sameacetic acid solution. Fractions were then collected, 5 ml. each.

The pattern obtained by gel filtration on Sephadex G-75 is illustratedin FIG. 5. Recovery was 76 percent. It was evidenced from the above'thatsaid acid carboxypeptidase is eluted far more rapidly than acidproteinase (Aspergillopeptidase A) whose molecular weight is 35,000,indicating that it is therefore an enzyme having higher molecularweight.

The enzyme preparation obtained above was lyophilized directly or aftersalting-out to obtain pure acid carboxypeptidase preparation.

Furthermore, disc-electrophoresis was performed on the above enzymepreparation at pH 8.0 [D. E. Williams, R. A. Reisfeld: Ann. New YorkAcad. Sci. 121, 373 (1964)], and said enzyme demonstrated that it washomogeneous electrophoretically. The purified said enzyme preparationappears to be homogeneous on ultracentrifugation, and the sedimentationconstant at zero concentration is calculated as 7.3 S.

We claim:

1. A process for the production of a new enzyme, acid carboxypeptidase,comprising the steps of a. cultivating an organism selected from thegroup consisting of: Aspergillus usamii R-0635 (ATCC-l433 l Asperfiillussaitoi R-38l3 (AT CC :14332 As er illus ni er RRL 330; Aspergzllus munR36 1 T C-l433 Aspergillus aureus R-4523; Aspergillus aureus R-65l2(ATCC-l4334); Aspergillus awamori R-3523; Aspergillus awamori [AM 2390(ATCC-l4335); Aspergillus nakazawai R-6822Y; Aspergillus oryzae var.magnasporus A- l-5; Aspergillus sojae KS; and b. recovering frommaterial selected from the culture broth and preparations therefrom acidcarboxypeptidase which reacts hydrolytically with the terminal carboxylgroup of proteins or peptides to give an amino acid and has an optimumpH of about 3.1 to 3.5 at a temperature of about 30 C. in acting on asubstrate selected from the group consisting ofbenzoxycarbonyl-L-tyrosyl-L-leucine,benzoxycarbonyl-L-glutamyI-L-tyrosine, andbenzoxycarbonyl-glycl-L-prolylL-leucyl-glycine contains no metal, has amolecular weight of about 122,000 and is inactive at 60 C. or more or apH of 7 or more.

